For several years we have been studying the molecular actions of the PTPN22 gene product, the lymphoid tyrosine phosphatase LYP, which is expressed only in white blood cells and acts as a gatekeeper for T lymphocyte activation. We reported what was to us the remarkable and seminal observation that a missense single-nucleotide polymorphism, C1858T in the PTPN22 gene, is associated with type 1 diabetes. It has been rewarding to note that a similar association was subsequently reported in several other human autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, juvenile idiopathic arthritis, and generalized vitiligo. Genetic studies by us and others have shown that the PTPN22 C1858T polymorphism is primarily associated with autoimmunity, findings that have been replicated in different populations. The molecular mechanism by which LYP tempers T lymphocyte activation through the T cell receptor (TCR) involves the formation of a complex between LYP and the negative regulatory kinase Csk. We were the first to report the remarkable molecular features that may well explain the autoimmune-predisposing behavior of the phosphatase variant LYP-W620 encoded by the PTPN22 T1858 allele: this variant cannot bind Csk, has gain-of-function characteristics, and shows increased enzymatic activity. As detailed in our preliminary data presented in this proposal, we recently found that in T cells, LYP undergoes reversible phosphorylation on tyrosine after TCR engagement, and that the autoimmune-associated LYPW620 shows reduced tyrosine phosphorylation in T cells. Thus, we hypothesize that reduced tyrosine phosphorylation of LYP mediates the action of the PTPN22 C1858T polymorphism in human autoimmunity. In this proposal, we will study tyrosine phosphorylation of LYP pursuing three specific aims which are: (1) to map LYP tyrosine phosphorylation sites and elucidate the functional effects of LYP tyrosine phosphorylation in TCR signaling; (2) to determine the mechanism underlying the reduced phosphorylation of the autoimmune-associated LYP-W620 variant; and (3) to create a knock-in mouse model and assess whether reduced phosphorylation of LYP affects incidence/severity of type 1 diabetes in mice. Our focus on LYP and its genetic variant provides a unique entrie into unlocking the mysteries of major human autoimmune disease mechanisms. Our studies will elucidate the mechanism of action of the LYP-R620W polymorphism at the molecular level that may aid treatment strategies, and develop a mouse model that can be used to study the variant in vivo, to test gene:gene and gene:environment interactions and to test therapeutic regimens in the future. PUBLIC HEALTH RELEVANCE This project focuses on the regulation of the lymphoid tyrosine phosphatase LYP, a protein involved with multiple human autoimmune disease, including type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, juvenile idiopathic arthritis, generalized vitiligo, and others. Carriers of a particular genetic variant of LYP are highly predisposed to autoimmunity. Our study is aimed at clarifying the effect of the autoimmune-predisposing variation on the functions of LYP. We predict that our results will open new avenues for designing therapies able to thwart the effects of the genetic variation, thus effectively preventing and/or treating autoimmunity in genetically predisposed individuals.